Persistent low-level variants in a subset of viral genes are highly predictive of poor outcome in immunocompromised patients with cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus is the most common and serious opportunistic infection after solid organ and haematopoietic stem cell transplantation. In this study, we used whole-genome cytomegalovirus data to investigate viral factors associated with the clinical outcome. METHODS: We sequenced cytomegalovirus samples from 16 immunocompromised paediatric patients with persistent viraemia. 8/16 patients died of complications due to cytomegalovirus infection. We also sequenced samples from 35 infected solid organ adult recipients of whom one died with cytomegalovirus infection. RESULTS: We showed that samples from both groups have fixed variants at resistance sites and mixed infections. NGS sequencing also revealed non-fixed variants at resistance sites in most of the patients who died (6/9). A machine learning approach identified 10 genes with non-fixed variants in these patients. These genes formed a viral signature which discriminated patients with cytomegalovirus infection who died from those that survived with high accuracy (AUC=0.96). Lymphocyte numbers for a subset of patients showed no recovery post-transplant in the patients who died. CONCLUSIONS: We hypothesise that the viral signature identified in this study may be a useful biomarker for poor response to antiviral drug treatment and indirectly for poor T cell function, potentially identifying early, those patients requiring non-pharmacological interventions.

Control of human cytomegalovirus replication by liver resident natural killer cells.

Natural killer cells are considered to be important for control of human cytomegalovirus- a major pathogen in immune suppressed transplant patients. Viral infection promotes the development of an adaptive phenotype in circulating natural killer cells that changes their anti-viral function. In contrast, less is understood how natural killer cells that reside in tissue respond to viral infection. Here we show natural killer cells resident in the liver have an altered phenotype in cytomegalovirus infected individuals and display increased anti-viral activity against multiple viruses in vitro and identify and characterise a subset of natural killer cells responsible for control. Crucially, livers containing natural killer cells with better capacity to control cytomegalovirus replication in vitro are less likely to experience viraemia post-transplant. Taken together, these data suggest that virally induced expansion of tissue resident natural killer cells in the donor organ can reduce the chance of viraemia post-transplant.

Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike.

The emergence of SARS-CoV-2 variants has exacerbated the COVID-19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N-terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine-tune spike; this may provide a mechanism for SARS-CoV-2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immune-driven antigenic variation and ongoing adaptation to a new host.

Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2.

The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARSCoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.

NK Cell Memory to Cytomegalovirus: Implications for Vaccine Development.

Natural killer (NK) cells are innate lymphoid cells that recognize and eliminate virally-infected and cancerous cells. Members of the innate immune system are not usually considered to mediate immune memory, but over the past decade evidence has emerged that NK cells can do this in several contexts. Of these, the best understood and most widely accepted is the response to cytomegaloviruses, with strong evidence for memory to murine cytomegalovirus (MCMV) and several lines of evidence suggesting that the same is likely to be true of human cytomegalovirus (HCMV). The importance of NK cells in the context of HCMV infection is underscored by the armory of NK immune evasion genes encoded by HCMV aimed at subverting the NK cell immune response. As such, ongoing studies that have utilized HCMV to investigate NK cell diversity and function have proven instructive. Here, we discuss our current understanding of NK cell memory to viral infection with a focus on the response to cytomegaloviruses. We will then discuss the implications that this will have for the development of a vaccine against HCMV with particular emphasis on how a strategy that can harness the innate immune system and NK cells could be crucial for the development of a vaccine against this high-priority pathogen.

Proteome-wide analysis of CD8+ T cell responses to EBV reveals differences between primary and persistent infection.

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor's individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response.

Redirection to the bone marrow improves T cell persistence and antitumor functions.

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

Double-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication.IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

UNLABELLED: The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE: Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways.